1474
Anal. Chem. 1989, 61 1474-7478
Analysis of Siderophores and Synthetic Hydroxamic Acids by High-Performance Liquid Chromatography with Amperometric Detection J e r e m y D. Glennon,* Michael R. Woulfe, A n d r e w T. Senior, and N u a l a NiChoileain
Department o f Chemistry, University College Cork, Cork, Ireland
Microbial siderophores are an important class of secreted metal sesquestering agent with applications ranging from Chelation therapy to precious metal recovery. The analysis of siderophores and of synthetic models of siderophores by highperformance Uquld chromatography (HPLC) is hampered by metal sequestering problems during the chromatography. The biocompatlbiiity of HPLC systems is examined here by comparison of the reversed-phase chromatographic behavior of a series of synthetic dihydroxamic acids, HONH-CO(CH,),-CO-NHOH ( n = 3-7) on metal-free and stainlesssteel HPLC systems. Problems associated with chelate formation on column are not encountered on the metal-free system. An applied voltage of +1.0 V (vs Ag/AgCi) Is optimum for amperometric detectlon and is used In conjunction with detectlon at 220 nm. For plmelk dlhydroxatnlc acid ( n = 5), under the given chromatographic conditions, the Iimk of detectlon is 5.8 X lo-' M using amperometrlc detection. The greater sensitivity and Selectivity achleveble with amperometric detection and illustrated in this work on dihydroxamic aclds can be exploited in the analysis of medically and industrially important hydroxamic acids. Desferoxamine, the commercially avaliable Chelation therapy drug, and Its Fe( I I I ) complex can be simultaneously analyzed by using amperometric detection with limits of detection down to 4.1 X I O 4 and 8.4 X lo-' MI respectively. The selective monitorlng of siderophores produced in liquid culture by genetlcally englneered stralns of Pseudomonas fluorescens is performed by direct InJectionof filtered supernatant samples.
Microbial metal mining is a term recently applied to the use of microorganisms for metal extraction and recovery ( I ) . This development in biotechnology is boosted by the availability of genetically engineered bacterial strains with enhanced metal sequestering abilities. Both the secreted agents and the biomass itself have found applications in extractive metallurgy and waste treatment. Microbial siderophores are an important class of secreted metal sequestering agent with applications in clinical medicine, biotechnology, industry, and analytical chemistry. Functional groups particularly responsible for the high selectivity of these agents for Fe(II1) include the hydroxamate and catecholate groups. The trihydroxamic acid, desferoxamine, is a commercially available siderophore used for the treatment of iron overload and aluminum intoxication ( 2 ) . The immobilization of siderophores onto solid supports provides resins for the recovery of trace metals from aqueous solution ( 3 ) . In particular, a polyhydroxamic acid resin was used in the recovery of uranium from seawater ( 4 ) . Hydroxamic acids have received considerable attention also as reagents in analytical chemistry for gravimetric analysis ( 5 ) and for the solvent extraction and spectrophotometric determination of metals (6). The reagents have also been shown to be useful in the analysis of trace metals by flow injection analysis (7) and high-performance liquid chromatography. 0003-2700/89/0361- 1474$01.50/0
Complexes of Zr(IV), Hf(IV), Fe(III), Nb(V), Al(III), and Sb(II1) with N-methylfurohydroxamic acid have been separated on a polymeric column with high chromatographic efficiency (8). The demand for sensitive and selective methods of analysis for these hydroxamic acid ligands has increased as a result of their wide applications. Methods that have been used include hydrolysis and derivatization techniques (9),paper electrophoresis ( I O ) , spectrophotometry (11),and thin-layer (12) and gas chromatographic analysis (13). A number of HPLC methods have been reported for a select group of clinically important hydroxamic acids, in particular for aromatic hydroxamic acids believed to be activated metabolites of carcinogenic arylamides. These include the analysis of aromatic hydroxamic acids in complex mixtures (14), N hydroxy derivatives of phenacetin, acetaminophen, and other hydroxamic acids as their Fe(II1) complexes (15) and, more recently, desferoxamine with its Fe(II1) and Al(II1) chelates (16,17). It is clear that there is an increasing need for efficient methods of analysis of these agents, whether they are products of biotechnology, chemical synthesis, the environment, or metabolism. The method of choice requires the added attribute of biocompatibility because of the strong metal binding properties of these compounds. The hydroxamic acids under investigation here are shown in Figure 1. Research in this laboratory has led to a convenient synthesis of a series of strongly chelating dihydroxamic acids, HOHN-CO-(CH2),-CO-NHOH ( n = 3-14), which were previously not readily available (18). These compounds are simple models for the naturally occurring siderophore, rhodotorulic acid (19). The reagents have a particularly strong affinity for Fe(II1) and are being investigated with respect to their biological activity, coordination chemistry (2O), and analytical applications. In this work the chromatographic behavior of the water-soluble acids on stainless steel and inert HPLC systems is investigated. Chromatographic conditions are optimized on a metal-free system and an improvement in sensitivity is demonstrated by the use of amperometric detection. The combination of amperometric detection with chromatography on an inert system offers considerable advantages in the general area of hydroxamic acid analysis. The method is applied to the detection of the important chelation drug desferoxamine (DFA) and to its Fe(II1) complex ferrioxamine (FA). The monitoring of siderophore production by Pseudomonas fluorescens can be carried out by direct injection of filtered supernatant samples. EXPERIMENTAL SECTION Instrumentation. Two HPLC systems were employed in this work. The first chromatographic analyses were carried out on a stainless steel chromatograph consisting of a Pye-Unicam LC3-XP pump, a Rheodyne Model 7125 sample injector (20 pL), and an LC3-UV detector. Separator columns used include a Chrompack Nucleosil C18 (25 cm X 4.0 mm i.d.) and a Waters pBondapak C18 column (25 cm X 4.6 mm id.). The inert HPLC system consisted of a Dionex analytical pump, an inert highpressure injection valve (10 pL), and a Dionex RPIC C18 10-pm 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 14, JULY 15, 1989 DHA HN-C-lCHRl,-C-NH
I
'1
HO 0
II I
0 OH
DFA NH~ICHslaN-ClCH~lrCON~lCHslaN-ClCHRlRCONHlCH~l~N-CCH~
I II
I II
I 1 I
HO
0
HO 0
HO 0
FA
Figure 1. Structures of di- and trihydroxamic acids.
Table 1. Relative Standard Deviations and Limits of Detection for Hydroxamic Acids, HOHN-CO-(CH2),-CO-NHOH and Siderophoresn
uv n
product
3
glutaric adipic pimelic suberic DFA FA
4
5 6
EC
mean % RSD
LOD, M x 10+6
mean % RDS
LOD,M x 10+6
1.1
6.1 7.4 8.4 15.6 1.8
1.7 2.4 2.3 3.2 3.6 3.8
3.2 4.9 5.8 1.2 0.04
0.8
0.9 1.4 2.7 3.4
0.6
0.8
Concentration ranges studied: DHA, 6.0 X 10" to 2.5 X lo4 M; DFA. 1.0x 10-5 to 1.0 x 10-4 M: FA. 5.0 x 104to 4.0 x 10-5 M. analytical column (25 cm X 6 mm id.). Dual detection was carried out by using UV detection at 220 nm in conjunction with an LC-17 amperometric detection system from Bioanalytical Systems Inc. dc amperometric detection was carried out with a Metrohm 626 Polarecord. An applied potential of + L O V (vs Ag/AgCl) was found to be optimum. A flow rate of 1.5 mL/min was used, unless otherwise stated. For stationary cell work a mini glassy carbon electrode, obtained from Metrohm (Herisau, Switzerland), was used in conjunction with a platinum wire auxiliary electrode and a Ag/AgC1(3 M KC1) reference. A Princteon Applied Research Model 364 polarographic analyzer was also employed. Infrared spectra were recorded as 1% KBr disks on a Perkin-Elmer Model 682 spectrophotometer. Proton NMR spectra were recorded in Me,SO-d6 on JEOL GX270, JOEL PS100, and Perkin-Elmer R12 60-MHz instruments. Synthesis and Characterization of Reagents. The two-step derivatization reaction of aliphatic dicarboxylic acids using N,N'-carbonyldiimidazole as previously reported from our laboratory was employed to produce the series of water-soluble reagents shown in Table I (18). Important practical advantages of this method are the mild conditions used, the stability of the diimidazolide intermediates, and the direct precipitation of the desired product from the reaction mixture. The products were characterized by infrared and proton NMR spectroscopy. The proton NMR spectrum obtained for pimelodihydroxamic acid ( n = 5) reveals the important characteristic resonances for the N-OH and N-H protons at 10.35 and 8.69 ppm (18). Bacterial Culture Supernatant Samples. A selected strain of Pseudomonas fluorescens was maintained on minimal asparagine agar consisting of sucrose (20 g), L-asparagine (2 g), K2HP04 (1 g), MgS0,.7H20 (0.5 g), and agar (15 g) in a liter of triply distilled water. Liquid medium contains all of the above constituents except agar. Colonies from agar plates were transferred to liquid media and the liquid cultures were grown overnight a t 30 OC, with aeration by vigorous shaking. Overnight cultures were then used to inoculate (1%inoculum) 100-mL volumes of sterile media in 500-mL Erlenmeyer flasks. After 12 h, bacteria were removed from the cultures by centrifugation at 10 000 rpm for
1475
10 min. The supernatants were collected and filtered prior to injection on the HPLC system. Reagents and Standard Solutions. All reagents were of analytical grade purity unless otherwise stated. Desferoxamine (DFA) was obtained from Ciba Geigy, as the commercially available drug "desferal mesylate". N,N'-Carbonyldiimidazole was obtained from Sigma Chemical Co. The chemicals used for mobile phase preparation were obtaned from BDH Chemicals (sodium chloride, sodium nitrate, acetic acid, citric acid, ethylenediaminetetraacetic acid, and sodium hydroxide). HPLC grade methanol was obtained from Rathburn Chemicals (Walkerburn, UK). Anal& ferric nitrate was used to prepare a 0.1 M Fe(II1) stock solution in 1% "0% Stock solutions (0.1 M) of the dihydroxamic acids listed in Table I were prepared by using the relevant mobile phase as sample solvent. Standard solutions were subsequently prepared from the stock in the concentration range 2.5 X W4to 1.0 X M. A reduction in the solubility of the longer chained dihydroxamic acids ( n = 6, 7) was observed for sample solvents with low methanol concentrations (Le.
